Process for producing a soluble rubella antigen

ABSTRACT

Purified soluble antigen, specific for rubella virus, is isolated from growth media of rubella-infected cell cultures by affinity and gel permeation chromatography and characterized, inter alia, by its specific activity. Antigen-sensitized particles are employed as immunoassay reagents in, for example, agglutination assays for detection and quantification of rubella antibodies in body fluids such as serum, spinal fluid and the like.

This is a division of application Ser. No. 773,231, filed Mar. 1, 1977.

BACKGROUND OF THE INVENTION

The present invention relates generally to materials and methods usefulin the detection of antibodies and particularly relates to a novel,soluble, rubella virus antigen. The antigen of the invention is employedto develop specific immunoassay reagents useful for rapid detection andquantification of rubella antibodies in test fluids. Materials andmethods of the present invention are useful in establishing theimmunological status of a patient, (e.g., a woman of child-bearing age)and are also of value in diagnostic programs.

Procedures commonly employed for determination of anti-rubellaantibodies in test fluids are based upon antibody inhibition of babychick erythrocyte hemagglutination by an insoluble rubella virusparticle. Among the essential steps of such procedures is the absorptionof test fluids with kaolin to effect removal of non-specific lipoproteininhibitors and absorption of the sera with baby chick erythrocytes toremove cross-reacting antibodies present in the fluid all prior totesting agglutination inhibition. Hemagglutination inhibition (HAI)assays of this type are relatively reliable but are time consumingbecause of the above-mentioned serum pre-treatment steps. Final testresults are ordinarily not available for at least about 5-7 hours aftertest fluid collection. Other techniques for detection of antibody torubella are summarized, e.g., in Meyer, H. M., et al., Am. J. Chin.Pathol., 57: 803-813 (1972).

Prior attempts have been made to secure a soluble rubella virus antigen,apart from the insoluble hemagglutinins used in HAI tests. The artdescribes identification of two major "soluble" antigens (designatedtheta and iota) but attempts to definitively isolate and characterizethese antigens from rubella-infected cell cultures have met with limitedsuccess and no soluble antigen heretofore isolated has been useful indeveloping an antigen-sensitized particle effective in detection andquantification of antibodies to rubella.

SUMMARY OF THE INVENTION

According to the present invention a soluble rubella virus antigen isisolated from media supporting growth of tissue culture cells infectedwith rubella virus. The antigen has a molecular weight of from about40,000 to about 60,000 daltons; is insoluble in 50% saturated ammoniumsulfate; and exhibits β mobility in immunoelectrophoresis.

More specifically, the novel antigen is characterized by forming asingle line precipitate with human serum reactive to rubella virus (asshown by hemagglutination inhibition tests). The antigen is furthercharacterized as having a specific rubella antigen activity (S.R.A.A.)of from about 500 to about 10,000.

The purified antigen is isolated by process steps including: affinitychromatography; gel permeation chromatography; and isolation on thebasis of relative reverse passive hemagglutination (RPHA) activity.

Immunological reagents of the invention are provided when the antigen isemployed to sensitize immunologically inert particulate materials suchas stabilized erythrocytes, bentonite, collodium, cholesterol crystals,quartz, synthetic resins, various kinds of synthetic latex, andliposomes prepared from phospholipids and sterols. Sensitized particlesare employed in direct agglutination assays wherein rubella antibodiespresent in a given test fluid sample are rapidly detected by observationof particle agglutination phenomena and quantified by standard dilutiontechniques. This passive agglutination method does not ordinarilyrequire removal from test fluid of non-specific inhibitors orantierythrocyte antibodies as do the HAI methods of the prior art.

Sensitized particles of the invention may also be employed inradioimmunoassay (RIA) and enzyme immunoassay (EIA) techniques. Further,the soluble antigen of the invention is expected to be useful inpractice of well known immunoprecipitation assay technique.

Advantages attending the use of the antigen and reagents and practice ofimmunological assay methods of the invention will be apparent uponconsideration of the following detailed description.

DETAILED DESCRIPTION

The soluble antigen of the invention is isolated from the culture mediumof rubella virus infected cells. Cell lines suitable for tissue culturegrowth to obtain the antigen may include Baby Hamster Kidney (BHK-21),Porcine Stabile Kidney (PS), Serum Institute Rabbit Cornea (SIRC) andothers well known in the art. In general, tissue cultures employed[according to the methods of Stewart, et al., N.E. Jour. Med. 276, No.10 pp. 554-7(1967)] for production of insoluble rubella hemagglutininsfor HAI tests are well suited for use according to this invention.

Isolation of the antigen proceeds by two-step chromatographic separationof growth medium components. As previously noted, the culture medium,preferably first concentrated by forced dialysis, is initially subjectedto affinity chromatographic separation by passage through a columnconsisting of a solid phase to which IgG, derived from human serum knownto contain antibodies reactive with rubella virus, has been conjugatedor covalently bonded. Preferred solid phase materials for the columninclude agarose beads.

After washing through unbound material, the antigen bound to the IgG iseluted with a suitable buffer. A glycine-sodium hydroxide buffer with apH in excess of 8 is preferred. Where particularly high pH buffers areemployed, neutralization of eluted materials may be advisable.

Separation of the antigen from higher molecular weight material whichmay bind to the affinity column and be eluted with the buffer isaccomplished by gel permeation chromatography involving, e.g., aSephadex G-150 column. The effluent from the column is monitored on a UVspectrophotometer at 280 nm and reverse passive hemagglutination bystandard techniques is employed to identify fractions containing thepurified antigen. The agglutination employs, e.g., erythroctyessensitized with human IgG from the same source as employed for preparingthe affinity column.

The antigen so obtained is insoluble in 50 percent ammonium sulfate;displays a sedimentation coefficient of approximately 3.4S; has anestimated molecular weight of about 40,000 to 60,000 as determined bySephadex G-150 chromatography; and exhibits β mobility inimmunoelectrophoresis.

The antigen is more particularly characterized by forming a single lineprecipitate with human serum reactive to rubella virus inhemagglutination inhibition tests. The antigen is also preciselycharacterized by having a specific rubella antigen activity (S.R.A.A.)of from about 500 to about 10,000. As employed herein, S.R.A.A. valuesare developed according to the following criteria. Any given crudetissue culture medium from growth of rubella-infected cells will displayabsorbancy at 280 nm. A typical crude medium from infected BHK-21 cellsdisplays an absorbancy of about 1.1 when compared to water. The crudemedium will also have a relatively fixed titer as determined by RPHA.Once again, a typical crude medium from infected BHK-21 cells willdisplay a titer of 1:32. The "total A₂₈₀ units" of material found in thecrude culture medium is defined as the volume (in ml) multiplied by theabsorbance at 280 nm. By dividing the reciprocal of the RPHA titer bythe total A₂₈₀ units, the S.R.A.A. is determined. S.R.A.A., therefore,equals the reciprocal of the RPHA titer divided by the total A₂₈₀ units.

The following illustrative examples relate to: (1) preparation of a"concentrated" cell culture medium containing the antigen of theinvention; (2) preparation of human IgG for use in affinitychromatography and reverse passive hemagglutination; (3) preparation ofthe affinity gel; (4) preparation of the gel permeation chromatographycolumn; (5) purification of the antigen from the "concentrated" medium;(6) preparation of rubella antigen-sensitized erythrocytes.

EXAMPLE I Preparation of Concentrated Medium Containing Antigen

BHK-21 cells were monolayered in 20 liter roller bottles and innoculatedwith Gilchrist strain rubella virus. After three to four days ofincubation, the medium was harvested and subjected to zonalcentrifugation and effluent is saved. This effluent is concentrated100-fold at 2°-8° C. in an Amicon DC-2 hollow fiber dialyzerconcentrator. The concentrated material is clarified by centrifugationat 9000 rpm for 30 minutes followed by ultracentrifugation at 29000 rpmfor 6 to 18 hours. The resulting concentrated material may be stored at-20° C.

EXAMPLE II Preparation of IgG

Human recalcified plasma with a rubella titer of approximately 1:640 isprecipitated with ammonium sulfate, dialyzed and purified according tothe following procedural sequence.

(1) Equal 150 ml volumes of saturated ammonium sulfate and humanrecalcified plasma which is rubella positive are admixed at a rate of 6to 10 ml/mixture while stirring with a magnetic stirrer at roomtemperature. The pH is adjusted to approximately 7.3 with sodiumhydroxide.

(2) The mixture is stirred for approximately one hour and theprecipitate form is collected by centrifugation at 9000 rpm for 30minutes at 2°-8° C.

(3) Centrifuged precipitate is added to dialysis tubing and dialyzedagainst two liters of 0.01 M K₂ HPO₄ /0.01 M KH₂ PO₄ buffer, pH of 8.0,with five changes of two liters each of the same buffer over 48 hours.

(4) The dialyzed material is recovered and is further purified throughuse of a Whatman DE52 Diethylaminoethyl Cellulose Microgranular(preswollen) Anion Exchanger and the IgG pool collected is subjected tofurther concentration, clarified by centrifugation and stored.

(6) The final yield of IgG recovered ranges from 150 mg to 160 mg per 70ml of whole serum.

EXAMPLE III Preparation of Affinity Gel

Sepharose 4b (Pharmacia) or any suitable agarose solid phase isactivated with cyanogen bromide, 97% (Aldrich) in acetonitrile andsubsequently coupled with human IgG (as prepared in Example II). Thecoupling of IgG to the solid phase is accomplished by practice of themethod of Cuatrecasas, J. Biol. Chem., 245: 3059-65 (1970) as modifiedin March, S.C., et al., Analyt. Biochem., 60: 149-52 (1974).

EXAMPLE IV Preparation of Gel Permeation Column

The gel permeation column for use in purifying antigen eluted from theaffinity column is prepared by the following procedure.

(1) Eighteen gm of Sephadex G-150 (Pharmacia) is added to 1 liter of0.05 M Tris-HCl in 0.15 M NaCl/0.02%NaN₃ pH 8.0 buffer, mixed, allowedto swell at room temperature for 3 days and degassed.

(2) The swollen gel slurry is added to a column to which the buffer hasbeen added and partially drained, with hydrostatic pressure at 3-5 cmduring the packing.

(3) The column is operated with use of a peristaltic pump, equilibratedwith two bed volumes of the buffer at a flow rate of 4.8 ml/cm² /hour,and tested for homogeneity with 6-8 ml of 0.2% Blue Dextran 2000,collecting 12 ml fractions.

EXAMPLE V Purification of Antigen from Concentrated Medium

Purification of soluble antigen from the concentrate of Example Iproceeds by (A) affinity chromatography and (B) gel permeationchromatography as follows:

A. Affinity Chromatography

(1) The affinity column of Example III is warmed to room temperature andthe buffer is drained to the top of the bed.

(2) The column is loaded with 3 bed volumes of ultracentrifuged"concentrate" of Example I at about 1 ml/minute flow rate.

(3) The column is washed with 5 bed volumes of 0.05 M Tris-HCl in 0.15 MNaCl-0.02% NaN₃ pH 8.0 buffer at 2-3 ml/minute.

(4) Following the wash, the column is eluted with 6 bed volumes of 0.1 Mglycine-NaOH in 0.15 M NaCl pH 12 buffer at about 1 ml/minute. Theeluted material is collected.

(5) With minimal delay, the eluted material is neutralized to pH 8.0 byadding 1 N HCl, dropwise, with constant stirring.

(6) The neutralized material is concentrated 5-fold of the original loadvolume in a single hollow fiber concentrator and clarified bycentrifugation.

B. Gel Permeation Chromatography

The material eluted from the affinity column is chromatographed on aSephadex G-150 column as prepared in Example IV. The fractions elutingin a volume expected to contain material with a molecular weight of40,000 to 60,000 daltons are collected and RPHA titers are determined.Fractions with a titer equal to or in excess of 1:6400 are pooled.S.R.A.A. values may be determined based on the A₂₈₀ value and RPHA titerof the pooled fractions. Typically, the antigen is concentrated to a 3to 8 ml volume from an original 80 to 100 liter volume of crude growthmedium and has a S.R.A.A. value of from 500 to 10,000.

EXAMPLE VI Rubella Antigen-Sensitized Erythrocytes

Human erythrocytes are stabilized according to the procedures disclosedin U.S. Pat. Nos. 3,714,345, 3,715,427 and/or 3,925,541; made up in 2.0ml, 10% suspensions, and centrifuged for 2 to 3 minutes at 500-1000 rpm.The buffer is decanted and the cells are resuspended in 2.0 ml of 0.01 Macetate-pH 4.0 buffer. 0.2 ml of aqueous chromic chloride solution (10mg CrCl.6H₂ O/ml) is added to the erythrocyte suspension. 0.05 to 0.50ml of antigen from Example 5 is added to the erythrocytes, thesuspension is incubated at 30°-32° C. for 2 hours with mixing at 30minute intervals. Sensitized erythrocytes are pelleted by centrifugationand the supernatant is discarded. The erythrocytes are washed twice byre-suspending in 0.1 M phosphate buffer and centrifuging as before. Thepellet is re-suspended in 0.1 M phosphate buffer in quantities providinga 0.125% (v/v) suspension of sensitized erythrocytes.

EXAMPLE VII

Sensitized erythrocytes, essentially according to Example VI, wereemployed to determine the antibody titers of random human blood donorserum samples and results were compared to titers obtained by HAItechniques. 1336 serum samples were tested and the correlationcoefficient (r) was determined as 0.99 by linear regression analysis.Using the sensitized erythrocytes there is no need to pre-absorb theserum samples to remove antibody cross-reacting with heterologouserythrocytes. Additionally it is unnecessary to pre-treat the serumsamples to remove non-specific lipoprotein inhibitors.

Numerous modifications and variations of the abovedescribed inventionwill occur to those skilled in the art. For example, the antigen may beemployed to sensitize immunologically inert particles of varying typeswell known in the art as useful in antigen-antibody detection schemes.In this regard, sensitized particles may be used in the detection ofantibody by agglutination techniques, by radioimmunoassay techniques, byfluorescent techniques, and by enzyme immunoassay techniques.Additionally, particles such as erythrocytes and liposomes may besensitized to provide an assay based on complement-mediated lysis.

What is claimed is:
 1. A process for producing a soluble rubellaantigen, said process comprising, in sequence:(a) subjecting growthmedia of a tissue culture of rubella virus infected cells tochromatographic separation through a column consisting of a solid phaseto which IgG, derived from human serum known to contain antibodiesreactive with rubella antigens, has been convalently coupled; (b)eluting from said column the rubella antigen materials bound to the IgGtherein with a buffer having a pH of about 12; (c) neutralizing therubella antigen materials to a pH of about 8; (d) subjecting theseparated eluted rubella antigen materials to gel permeationchromatography to achieve material separation on the basis of molecularsize; (e) collecting the fractions containing said materials having amolecular weight of about 40,000 to about 60,000 daltons; (f) isolatingrubella antigen from said sized, separated eluted materials on the basisof relative reverse passive hemagglutination activity.
 2. The process ofclaim 1 wherein, in step (a), the solid phase consists of agarose beads.